The present invention relates to a process for the manufacture of polyguluronic acids having degrees of polymerization less than 20 and substantially free of mannuronic acid contamination. It also relates to a process for the manufacture of concentrated alginic acid solutions for use in the formation of such polyguluronic acids.
Polyguluronic acids, because of their high affinity for calcium ions, are expected to have utility as scale inhibitors and scale deposit removers. The biodegradability of polyguluronic acids makes them particularly valuable with respect to environmental acceptability and waste disposal. Additionally, polyguluronic acid derivatives, in which a hydrophobic polymer is covalently attached to the reducing terminus of the polyguluronic acid, are useful as dispersants in pigment dispersed aqueous ink compositions for use in ink jet printing. Furthermore, polyguluronic acids, having low degrees of polymerization, have been shown to exhibit root-growth promoting activity in barley (M. Natsume et al., xe2x80x9cIsolation and Characterization of Alginate-derived Oligosaccharides with Root-Growth Promoting Activities,xe2x80x9d Carbohydrate Research, 258, 187-197 (1994)). They have also been shown to exhibit germination and shoot-elongation promoting activity in unhulled rice and tobacco callus (Y. Yonemoto et al., xe2x80x9cPromotion of Germination and Shoot Elongation of Some Plants by Alginate Oligomers Prepared with Bacterial Alginate Lyase,xe2x80x9d Journal of Fermentation and Bioengineering, 75, 68-70 (1993)). Based on studies of other polyuronic acids, low molecular weight polyguluronic acids might also be expected to exhibit antiviral, antitumoral, and plant-defense stimulating activities.
Alginic acids, from which polyguluronic acids can be obtained, are unbranched polymers of 1xe2x86x924 linked xcex1-L-guluronic acid (G) and xcex2-D-mannuronic acid (M) of varying proportions and sequence. A typical structure of an alginic acid molecule may be represented schematically as follows:
As can be seen from the above structure, the distribution of monomers in alginates is not random and there is no regular repeat unit. Alginic acids are best described as block copolymers in which there are polyguluronic acid sequences (G-blocks), polymannuronic acid sequences (M-blocks), and sequences containing random arrangements of both guluronic acid and mannuronic acid (MG-blocks).
It is well known that alginates, which are salts of alginic acid, can be hydrolyzed and that the hydrolysis products can be separated to give two predominantly homopolymeric fractions, polyguluronic acid and polymannuronic acid. The most often cited procedure for the preparation of the sodium salt of polyguluronic acid is a heterogeneous acidic hydrolysis method disclosed in A. Haug et al., xe2x80x9cStudies on the Sequence of Uronic Acid Residues in Alginic Acid,xe2x80x9d Acta Chemica Scandinavica, 21, 691-704 (1967). The acidic hydrolysis described therein requires that one part of sodium alginate be suspended in twenty parts of 0.3 M hydrochloric acid solution. Because alginic acid is insoluble in the strongly acidic solution, the hydrolysis is a heterogeneous reaction. The heterogeneous mixture is heated for 10+hours at 100xc2x0 C. and then the solid is separated from the acidic solution by centrifugation or filtration. After the collected solid is dissolved in water by neutralizing with dilute sodium hydroxide solution, twenty parts of 0.3 M hydrochloric acid solution are added to the solution resulting in reprecipitation of the partially hydrolyzed alginic acid. The resulting heterogeneous mixture is heated for an additional 10+hours at 100xc2x0 C. and the solid is again separated from the acidic solution by centrifugation or filtration. The collected solid is dissolved in water by neutralizing with dilute sodium hydroxide solution and then sodium chloride and water are added to yield a solution which is 0.5 wt % alginic acid salt and 0.1 M sodium chloride. An approximately equal volume of 0.025 M hydrochloric acid solution is added to the alginic acid salt solution until a pH value of 2.85 is obtained. The precipitated solid is separated from the acidic solution by centrifugation or filtration. The isolated solid is dissolved in water by neutralizing with dilute sodium hydroxide solution and then precipitated with excess ethanol. The precipitated solid is washed with ethanol, washed with ether, and dried. The sodium salt of the polyguluronic acid prepared by this heterogeneous acidic hydrolysis method has an average degree of polymerization between 15 and 20. The mannuronic acid content is between 5 and 15% and the yield of product is between 15 and 20%.
During the course of the first step of the heterogeneous acidic hydrolysis, approximately 30% of the alginate goes into solution. An additional 15% of the original alginate goes into solution during the course of the second step. The insoluble fraction which is isolated after both steps contains both polyguluronic acid and polymannuronic acid. In the acidification of the dilute solution containing both sodium salts of polyguluronic acid and polymannuronic acid, polyguluronic acid is selectively precipitated.
Although the heterogeneous acidic hydrolysis method of A. Haug et al. is useful for laboratory scale preparations of polyguluronic acid, it would be difficult to implement on a larger scale, such as that which would be used in industrial production. This is because in the separation step of that method, the concentration of alginic acid is only a very dilute 0.25 wt %. Additionally, the method has multiple steps and is complicated.
In the same reference as that describing the heterogeneous acidic hydrolysis method, a homogeneous acidic hydrolysis procedure is also reported. In that procedure, a 1 wt % sodium alginate solution is mixed with an equal volume of a 0.025 M citrate buffer solution such that a combined solution having a pH value of 3.6 is obtained. The solution is boiled at reflux for 5+hours. Although a method for isolating polyguluronic acids is not reported, presumably a method similar to that used in the heterogeneous acidic hydrolysis method can be used.
Although the homogeneous acidic hydrolysis method of A. Haug et al. may be useful for laboratory scale preparations of polyguluronic acid, it also would be difficult to implement on a larger scale. In that method, the concentration of sodium alginate in the hydrolysis step is only a very dilute 0.5 wt %. Although one can easily conceive of increasing the concentration of sodium alginate, in practice this cannot be done. At concentrations slightly greater than 0.5 wt %, sodium alginate does not remain soluble throughout the hydrolysis reaction in the range of pH values at which the acidic hydrolysis is effected. The species which precipitate in the course of the hydrolysis are incompletely hydrolyzed and, although rich in guluronic acid, they contain significant amounts of mannuronic acid. Furthermore, the incompletely hydrolyzed species have degrees of polymerization of greater than 20. Because the homogeneous acidichydrolysis conditions are milder than those described above in the heterogeneous acidic hydrolysis procedure, the incompletely hydrolyzed species are not further hydrolyzed after precipitating from solution.
Another means of obtaining low molecular weight hydrolysis products of alginic acid is disclosed in a published study of the hydrolytic degradability of partially dicarboxylated alginic acid (xe2x80x9cBiodegradability, Hydrolytic Degradability, and Builder Performance in Detergent Formulations of Partially dicarboxylated Alginic Acid,xe2x80x9d Journal of Environmental Polymer Degradation, 4, 113-121 (1996)). In that paper (which is hereby incorporated herein by reference), partially dicarboxylated alginic acid is prepared and then hydrolyzed to low molecular weight products. The hydrolysis products were only characterized by gel-permeation chromatography and, thus, their actual identity is unknown. However, it is reasonable to assume that the products were mixtures of the homopolymeric fractions, polyguluronic acids and polymannuronic acids. If one wanted to isolate the polyguluronic acids from those mixtures, presumably a procedure similar to that used in the heterogeneous acidic hydrolysis method of A. Haug et al. could be used.
The above described low molecular weight hydrolysis products of dicarboxylated alginic acid were obtained by using a three step process starting with alginic acid. In the first step, alginic acid was oxidized to partially diformylated alginic acid using a less than a stoichiometric amount of sodium periodate as the oxidant. The product was isolated and purified by dialysis. In the second step, the partially diformylated alginic acid was oxidized to partially dicarboxylated alginic acid using an excess of sodium chlorite as the oxidant. Again, the product was isolated and purified by dialysis. In the third step, the partially dicarboxylated alginic acid was dissolved in a buffer solution, having a pH value of 4, and incubated at 30xc2x0 C. for periods of time up to 30 days. These same first two oxidation steps also have been reported in xe2x80x9cAn Apparent 4C1xe2x86x921C4 Conformational Transition in Periodate-Oxidized Alginate,. Induced by Changes in pH and Ionic Strength: The Anomeric Effect in Glycuronans,xe2x80x9d Carbohydrate Research, 71, C9-C12 (1979). In that paper, aqueous bromine was used instead of sodium chlorite in the second oxidation step.
Although the above described three step process, in which partially dicarboxylated alginic acid is an intermediate product, may be useful for laboratory scale preparations of polyguluronic acid, it is not optimal for industrial production because the method has multiple steps and is complicated. Furthermore, it requires two different oxidants.
Thus, there remains a need for a method of manufacturing polyguluronic acids which can be carried out on an industrial scale.
It is an object of the present invention to provide a practical process for the manufacture of polyguluronic acids having a degree of polymerization less than 20 and substantially free of mannuronic acid contamination.
It is another object of the invention to provide a hydrolysis process using a solution of alginic acid or alginic salt in which the concentration of alginic acid or alginic salt is equal to or greater than 5 wt % throughout the process.
It is a further object of the invention to provide solutions of alginic acid or alginic salts for use in such hydrolysis process.
These and other objects of the invention may be achieved by virtue of the inventor""s discovery that periodic acid H5IO6 and periodate salts, which include but are not limited to sodium periodate (metaperiodate), NaIO4, sodium paraperiodate, Na3H2IO6, and potassium periodate, KIO4, will effect oxidation of concentrated solutions of alginic acid such that intermediate products which are easily hydrolyzed are obtained. It has been found that free iodine, and not iodates, can be made the major iodine containing product of the oxidation reaction. The implication of this finding is that multiple oxidation steps are being effected by one oxidant. Also, it has been found that 5 wt % or greater solutions of alginic acid, for the purpose of periodate oxidation, can be obtained by using lithium hydroxide to neutralize the alginic acid. These combined findings form a basis of the present invention.
According to the invention there is provided a method of treating alginic acid to produce polyguluronic acids, said method comprising the steps of:
(a) mixing the alginic acid with water and lithium hydroxide in respective amounts sufficient to form a reaction mixture that is an acidic solution comprising at least 5 wt % of the alginic acid;
(b) treating the reaction mixture, with addition of an oxidizing agent selected from the group consisting of a periodic acid and a periodate salt, to effect an oxidation of said alginic acid that results in formation of (i) an oxidized product that, upon acidifying and heating, hydrolyzes into a plurality of components including polyguluronic acids that are substantially free of mannuronic acid contamination, with the polyguluronic acids having an average degree of polymerization of less than 20; and (ii) free iodine;
(c) separating the free iodine from the reaction mixture;
(d) acidifying and heating the reaction mixture to hydrolyze the oxidized product into the plurality of components;
(e) adjusting the pH of the reaction mixture to form a precipitate consisting essentially of the polyguluronic acids; and
(f) recovering the precipitate.
In a preferred embodiment of the invention, a sufficient amount of the lithium hydroxide is mixed with the water and alginic acid in step (a) to form the acidic solution with a pH value of between 3.8-5.0. In another preferred embodiment, the oxidizing agent is added in step (b) in a less than stoichiometric amount. Preferably, the less than stoichiometric amount of the oxidizing agent is greater than 5 mole % and less than 50 mole %.
In accordance with the invention, the oxidizing agent may comprise a periodate salt selected from the group consisting of sodium periodate (NaIO4), sodium paraperiodate (Na3H2IO6) and potassium periodate (KIO4). The alginic acid may be oxidized in step (b) in an oxidation reaction that comprises an initial exothermic stage and a completion stage, with the method comprising heating the reaction mixture after the initial exothermic stage to effect a completion of the oxidation reaction. The method may also comprise cooling the reaction mixture during said initial exothermic stage.
In accordance with other preferred embodiments, the reaction mixture is acidified to a pH value of less than or equal to 3.5 in step (d). The acidified reaction mixture may be heated in step (d) at a temperature greater than or equal to 80xc2x0 C. The polyguluronic acids may be selectively precipitated in step (e) by adjusting the pH of the reaction mixture to a pH value greater than or equal to 3.0 and less than or equal to 3.4. The alginic acid in step (a) may have a weight average molecular weight of less than or equal to 50,000 g/mole. The precipitate in step (e) preferably comprises less than 8 wt % of mannuronic acid, and more preferably comprises less than 5 wt % of mannuronic acid.
There is also provided in accordance with another embodiment of the invention a method of treating alginic acid to produce a concentrated solution for use in producing polyguluronic acids, and concentrated solutions produced by such method. The method in accordance with this embodiment comprises:
(a) providing an alginic acid that comprises guluronic acid monomers and mannuronic acid monomers; and
(b) mixing the alginic acid with water and lithium hydroxide in respective amounts sufficient to produce the concentrated solution having a pH between 3.8-5.0 and containing at least 5 wt % of the alginic acid. In a preferred aspect of this embodiment, the guluronic acid monomers are present in the alginic acid with respect to the mannuronic acid monomers in a ratio of greater than about 1:1 and less than about 2:1. In another preferred aspect, the method may further comprise the steps of:
(c) treating the concentrated solution by addition of an oxidizing agent selected from the group consisting of a periodic acid and a periodate salt, to effect an oxidation of said alginic acid that results in formation of (i) an oxidized product that, upon acidifying and heating, hydrolyzes into a plurality of components including polyguluronic acids that are substantially free of mannuronic acid contamination, with the polyguluronic acids having an average degree of polymerization of less than 20; and (ii) free iodine; and
(d) separating the free iodine from the treated solution.